RESUMO
A fim de se avaliar o uso do óleo essencial de Croton nepetifolius Baill (OECn) na penetrabilidade cervical em ovelhas mestiças, 40 ovelhas foram distribuídas ao acaso em quatro grupos (n=10): controle, misoprostol, OECn50 e OECn100 (50 e 100µg do OECn, respectivamente). Após a sincronização do estro, utilizando CIDR e eCG (200UI), a profundidade de penetração da cérvix foi mensurada utilizando-se uma pipeta de inseminação artificial de bovino graduada, no período de zero até 72h após a retirada do CIDR. Os resultados foram expressos em média ± erro-padrão, submetidos à ANOVA seguida do teste de Tukey, enquanto os dados, em porcentagem, foram submetidos aos testes de Fisher ouqui-quadrado. Nenhuma diferença significativa (P>0,05) foi encontrada quanto ao grau de penetrabilidade cervical. Quanto ao tempo de passagem, os grupos misoprostol e OECn100 apresentaram um menor tempo de penetrabilidade às 60h(1,7±0,6 e 1,5±0,6min, respectivamente), quando comparados ao grupo controle (4,1±0,6min), que não diferiu significativamente do grupo OECn50 (2,3±0,6min). Portanto, o óleo essencial de Croton nepetifolius Baill pode ser utilizado para encurtar o tempo de penetrabilidade cervical em ovelhas submetidas à sincronização estral.(AU)
In order to evaluate the use of the essential oil of Croton nepetifolius Baill (EOCn) on cervical penetration in crossbred ewes, 40 ewes were randomly allocated into four groups (n= 10): CONTROL, MISOPROSTOL, EOCn50 and EOCn100 (50 e 100µg of the EOCn, respectively). After estrus synchronization, using CIDR and eCG (200IU), depth of cervical penetration was measured using artificial insemination gun for bovine species which was graduated and used from 0 to 72h after CIDR removal. Results were expressed as mean ± standard error mean, submitted to ANOVA and Tukey test while data in percentage were submitted to Fisher or Chi-Square test. No significant difference (P> 0.05) was observed at grade of cervical penetration. Concerning trespassing time, MISOPROTOL and EOCn100 groups presented a lower trespassing time at 60h (1.7±0.6 and 1.5±0.6min, respectively) than CONTROL group (4.1±0.6min), which did not differ significantly from EOCn50 (2.3±0.6min) group. Therefore, the essential oil of Croton nepetifolius Baill can be used to shorten the cervical penetration time in estrus synchronized ewes.(AU)
Assuntos
Animais , Feminino , Gravidez , Estro , Ovinos , Euphorbiaceae , Parto , Sincronização do EstroRESUMO
BACKGROUND: Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. METHODS: Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosphorylation (pFGFR) levels and M1R-FGFR1 heteroreceptor complexes, respectively. RESULTS: Oxotremorine-M, a non-selective mAChRs agonist, was able to transactivate FGFR and this transactivation was blocked by Src inhibitors. Oxotremorine-M treatment produced a significant increase in the primary neurite outgrowth that was blocked by pre-treatment with the pFGFR inhibitor SU5402 and Src inhibitors. This trophic effect was almost similar to that induced by fibroblast growth factor-2 (FGF-2). By using atropine as nonselective mAChRs or pirenzepine as selective antagonist for M1 receptor (M1R) we could show that mAChRs are involved in modulating the pFGFRs. Using PLA, M1R-FGFR1 heteroreceptor complexes were identified in the hippocampus and cerebral cortex. CONCLUSION: The current findings, by showing functional mAChR-FGFR interactions, will contribute to advance the understanding of the mechanisms involved in the actions of cholinergic drugs on neuronal plasticity. GENERAL SIGNIFICANT: Data may help to develop novel therapeutic strategies not only for neurodegenerative diseases but also for depression-induced atrophy of hippocampal neurons.
Assuntos
Hipocampo/metabolismo , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Muscarínico M1/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hipocampo/efeitos dos fármacos , Masculino , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The lack of dystrophin in mdx mice leads to cycles of muscle degeneration and regeneration processes. Various strategies have been proposed in order to reduce the muscle-wasting component of muscular dystrophy, including implementation of an exercise programme. The aim of this study was to examine how low-intensity endurance exercise affects the degeneration-regeneration process in dystrophic muscle of male mdx mice. Mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 days/week for 6 weeks. Histomorphological analysis showed a significant reduction of measured inflammatory-necrotic areas in both gastrocnemius and quadriceps muscle of exercised mdx mice as compared to matched sedentary mdx mice. The degenerative-regenerative process was also evaluated by examining the protein levels of connexin 39 (Cx39), a specific gene expressed in injured muscles. Cx39 was not detected in sedentary wild type mice, whereas it was found markedly increased in sedentary mdx mice, revealing active muscle degeneration-regeneration process. These Cx39 protein levels were significantly reduced in muscles of mdx mice exercised for 30 and 40 days, revealing together with histomorphological analysis a strong reduction of degeneration process in mice subjected to low-intensity endurance exercise. Muscles of exercised mdx mice did not show significant changes in force and fatigue resistance as compared to sedentary mdx mice. Overall in this study we found that specific low-intensity endurance exercise induces a beneficial effect probably by reducing the degeneration of dystrophic muscle.
Assuntos
Terapia por Exercício/métodos , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/terapia , Condicionamento Físico Animal/fisiologia , Regeneração/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Conexinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fadiga Muscular/fisiologia , Força Muscular/fisiologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/patologia , Distrofia Muscular Animal/fisiopatologia , Condicionamento Físico Animal/métodos , Resistência Física , Distribuição AleatóriaRESUMO
A number of in vitro and in vivo studies using selective agonists have indicated a neuroprotective role for group-II metabotropic glutamate (mGlu2/3) receptors in various models of neuronal injury. Although an interplay among neurotrophic factors and mGlu2/3 receptors signalling system has been suggested as possible mechanism involved on neuroprotection, at present poor information are available concerning the in vivo regulation by mGlu2/3 receptors activation of specific neurotrophic factors. By using in situ hybridization and western blotting methods the aim of present study was to analyse the potential regulatory role of selective mGluR2/3 agonist LY379268 treatment on brain derived neurotrophic factor (BDNF) expression in the mouse brain. The treatment with LY379268 evidenced a significant upregulation of BDNF mRNA levels in the cerebral cortex and in the hippocampal formation with a peak at 3 h from treatment and its disappearance already at 6 h from treatment. An analysis of dose-effect curve revealed that LY379268 may significantly enhance BDNF mRNA expression already at dose of 0.250 mg/kg b.w. The upregulation of BDNF mRNA expression was followed by a significant increase of BDNF protein levels at 24 h from LY379268 treatment. These effects of LY379268 treatment on BDNF expression were restricted to neuronal cells and were blocked by the new selective mGlu2/3 receptor antagonist LY341495, suggesting a receptor specificity. Taken together these findings suggest that several previous observed neuroprotective and trophic actions of mGluR2/3 agonists treatment may be mediated, at least in the cerebral cortex and hippocampal formation, by upregulation of BDNF expression.
Assuntos
Aminoácidos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Aminoácidos/administração & dosagem , Animais , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/metabolismo , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Xantenos/farmacologiaRESUMO
Neurogenesis occurs in two regions of the adult brain, namely, the subventricular zone (SVZ) throughout the wall of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampal formation. Adult neurogenesis requires several neurotrophic factors to sustain and regulate the proliferation and differentiation of the adult stem cell population. In the present review, we examine the cellular and functional aspects of a trophic system mediated by fibroblast growth factor-2 (FGF-2) and its receptors (FGFRs) related to neurogenesis in the SVZ and SGZ of the adult rat brain. In the SVZ, FGF-2 is expressed in GFAP-positive cells of SVZ but is not present in proliferating precursor cells, which instead express FGFR-1 and FGFR-2, but not FGFR-3 mRNA, although expressed in the SVZ, and FGFR-4. Therefore, it seems that in the SVZ FGF-2 may be released by GFAP-positive cells, different from the precursor cell lineage, and via volume transmission it reaches the proliferating precursor cells. FGFR-1 mRNA is also expressed in the SGZ and is localized in BrdU-labeled precursor cells, whereas FGFR-2 and FGFR-3 mRNA, although expressed in the SGZ, are not located within proliferating precursor cells. An aged-related decline of proliferating precursor cells in the SVZ and DG of old rats has been well documented, and there is the suggestion that in part it could be the consequence of alterations in growth factor expression levels. Thus, the old precursors may respond to growth factors, suggesting that during aging the basic components for neuronal precursor cell proliferation are retained and the capacity to increase neurogenesis after appropriate stimulation is still preserved. In conclusion, the trophic system mediated by FGF-2 and its receptors contributes to create an important micro-environmental niche that promotes neurogenesis in the adult and aged brain.
Assuntos
Encéfalo/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neurogênese/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Envelhecimento , Animais , Ventrículos Cerebrais/fisiologia , Humanos , Transdução de SinaisRESUMO
In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers or to repair the damaged one. The aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) 37, Cx39, Cx40, Cx43, and Cx45 expression in the adult regenerating skeletal muscle in response to crush injury. Within 24 h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24 h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesion-forming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24 h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers.
Assuntos
Conexinas/metabolismo , Músculo Esquelético/metabolismo , Regeneração , Animais , Agregação Celular , Fusão Celular , Proliferação de Células , Conexina 30 , Conexina 43/metabolismo , Conexinas/genética , Constrição , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Masculino , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Músculo Esquelético/cirurgia , Neovascularização Fisiológica , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regeneração/genética , Fatores de Tempo , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
Over the past years, evidence has accumulated that stem cells are present in the adult brain, and generate neurons and/or glia from two active germinal zones: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This study shows that acute intermittent nicotine treatment significantly enhances neuronal precursor cell proliferation in the SVZ of adult rat brain, but not in the SGZ of the hippocampus, and pre-treatment with mecamylamine, a nonselective nAChR antagonist, blocks the enhanced precursor proliferation by nicotine. This effect is supported by up-regulation of fibroblast growth factor-2 (FGF-2) mRNA in the SVZ and the expression of its receptor FGFR-1 in cells of SVZ showing precursor cells profile. It is also demonstrated that the nicotine effect on neuronal precursor proliferation is mediated by FGF-2 via fibroblast growth factor receptor 1 (FGFR-1) activation by showing that i.c.v. pre-treatment with anti-FGF-2 antibodies or with FGFR-1 inhibitor 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402) blocks nicotine-induced precursor cell proliferation. This nicotine enhancement of neuronal precursor cell proliferation was not accompanied by an increase in the number of apoptotic cells. Taken together the present findings revealed the existence in the SVZ of the adult rat brain of a trophic mechanism mediated by FGF-2 and its receptor and regulated by nAchR activation. This possibility of in vivo regulation of neurogenesis in the adult brain by exogenous factors may aid to develop treatments stimulating neurogenesis with potential therapeutic implications.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/genética , Ventrículos Laterais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nicotina/farmacologia , Células-Tronco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Nicotina/uso terapêutico , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/uso terapêutico , Antagonistas Nicotínicos/farmacologia , Pirróis/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
In the present work we reviewed recent advances concerning neuroprotective/neurotrophic effects of acute or chronic nicotine exposure, and the signalling pathways mediating these effects, including mechanisms implicated in nicotine addiction and nAChR desensitization. Experimental and clinical data largely indicate long-lasting effects of nicotine and nicotinic agonists that imply a neuroprotective/neurotrophic role of nAChR activation, involving mainly alpha7 and alpha4beta2 nAChR subtypes, as evidenced using selective nAChR agonists. Compounds interacting with neuronal nAChRs have the potential to be neuroprotective and treatment with nAChR agonists elicits long-lasting neurotrophic effects, e.g. improvement of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Nicotine addiction, which is mediated by interaction with nACh receptors, is believed to involve the modification of signalling cascades that modulate synaptic plasticity and gene expression. Desensitization, in addition to protecting cells from uncontrolled excitation, is recently considered as a form of signal plasticity. nAChR can generate these longe-lasting effects by elaboration of complex intracellular signals that mediate medium to long-term events crucial for neuronal maintenance, survival and regeneration. Although a comprehensive survey of the gene-based molecular mechanisms that underlie nicotine effects has yet not been performed a growing amount of data is beginning to improve our understanding of signalling mechanisms that lead to neurotrophic/neuroprotective responses. Evidence for an involvement of the fibroblast growth factor-2 gene in nAChR mechanisms mediating neuronal survival, trophism and plasticity has been obtained. However, more work is needed to establish the mechanisms involved in the effects of nicotinic receptor subtype activation from cognition-enhancing and neurotrophic effects to smoking behaviour and to determine more precisely the therapeutic objectives in potential nicotinic drug treatments of neurodegenerative diseases.
Assuntos
Encéfalo/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Humanos , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/uso terapêutico , Agonistas Nicotínicos/química , Agonistas Nicotínicos/uso terapêutico , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tabagismo/metabolismo , Tabagismo/fisiopatologiaRESUMO
Several studies in different in vitro and in vivo models have demonstrated neuroprotective effects of nicotinic receptor agonists and indirect trophic actions of nicotine on brain are suggested from observations describing nicotine as a cognitive enhancer by increasing vigilance and improving learning and memory. While an increasing number of studies have given evidence of neuroprotective and neurotrophic effects of nicotine treatment, the molecular mechanism mediating the neurotrophic effects of nicotine are not fully understood. Previously in an analysis of several neurotrophic factors as possible mediators of nicotine-induced neuroprotection and/or neurotrophic effects we could reveal that an acute intermittent nicotine treatment increases fibroblast growth factor-2 mRNA and protein in several brain regions of rat brain. Even if other studies have demonstrated in different paradigms that nicotine administration modulates expression level of a variety of genes, there is still a lack of indication which candidate genes, involved in neuroprotective responses are modulated by nicotine. In the present work we have used a microarray assay to further find and characterize new genes responsive to acute intermittent nicotine treatment and linked to neuroprotection. Therefore, we used Rat Genome U34A Affymetrix GeneChip arrays containing about 8800 probe sets to characterize transcriptional responses in the rat parietal cortex after acute intermittent nicotine treatment. We focused our attention to expression of transcription factors and several of them were up- or down-regulated by nicotine, among these Nr4a1 (Nurr77), Egr-1 and Egr-2. In situ hybridization was used to corroborate the microarray data and to reveal further spatial and temporal patterns of these nicotine induced genes. Taken together the present results identified several novel candidate genes modified by acute intermittent nicotine exposure and as such potentially involved in neuroprotective-neurotrophic actions.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiologia , Perfilação da Expressão Gênica , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Fatores de Transcrição/genética , Animais , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos WistarRESUMO
We report a detailed analysis of the expression pattern of the recently identified rat connexin gene, named rat connexin 39 (rCx39), both during embryonic development and in adult life. Qualitative and quantitative reverse transcription/polymerase chain reaction analysis showed intense expression of rCx39 restricted to differentiating skeletal muscles, with a peak of expression detected at 18 days of embryonic life, followed by a rapid decline to undetectable levels within the first week of postnatal life. A combination of the in situ hybridization technique for the detection of rCx39 mRNA and immunohistochemistry for myogenin, a myoblast-specific marker, allowed us to establish that the mRNA for this connexin was expressed in myogenin-positive myoblasts and early myotubes but disappeared in mature myotubes. Moreover, in adult animals, rCx39 mRNA was expressed in myogenic cells involved in skeletal myofiber regeneration following a crush injury. This is the first case of a connexin being mainly expressed in the myogenic cell lineage. The information presented should pave the way to novel molecular approaches in studies on the role of connexin-based gap-junctional communication in skeletal muscle differentiation and regeneration.
Assuntos
Conexinas/genética , Conexinas/metabolismo , Hibridização In Situ , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/embriologia , Mioblastos Esqueléticos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem da Célula , Conexinas/química , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
Glial connexins (Cxs) make an extensively interconnected functional syncytium created by a network of gap junctions between astrocytes and oligodendrocytes. Among Cxs expressed in the brain, Cx30 is expressed in grey matter astrocytes, as shown at the protein level by immunoistochemistry. In the present study we aimed to perform a detailed study of the regional distribution of Cx30 mRNA in the adult and postnatal developing rat brain, analyzing its expression by in situ hybridization, and determining its cell type localization by double labeling. Recently, it has been suggested that neuronal activity may control the level of intercellular communication between astrocytes through gap junctions channels. Thus, a second aim of the present study was to investigate the short-term effects of kainate-induced seizures on Cx30 expression. The results showed that, in basal condition, Cx30 was expressed only in grey matter astrocytes with distinct regional patterns in developing and adult brain. Kainate treatment induced strong and region-specific changes of astroglial Cx30 mRNA levels and expression of Cx30 mRNA in neuronal cells undergoing cell death, suggesting a direct or indirect involvement of this connexin in the neuronal apoptotic process.
Assuntos
Apoptose/genética , Astrócitos/metabolismo , Encéfalo/metabolismo , Conexinas/genética , Expressão Gênica/genética , Neurônios/fisiologia , Estado Epiléptico/genética , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Comunicação Celular/genética , Agonistas de Aminoácidos Excitatórios , Imuno-Histoquímica , Ácido Caínico , Masculino , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/fisiopatologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/fisiopatologia , Regulação para Cima/genéticaRESUMO
Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.
Assuntos
Fadiga Muscular/fisiologia , Músculo Esquelético/fisiologia , Fatores de Crescimento Neural/genética , Junção Neuromuscular/fisiologia , Acetilcolinesterase/metabolismo , Fatores Etários , Animais , Eletromiografia , Camundongos , Camundongos Knockout , Neurônios Motores/fisiologia , Contração Muscular/fisiologia , Fibras Musculares de Contração Lenta/enzimologia , Músculo Esquelético/citologia , Músculo Esquelético/inervação , Receptores Colinérgicos/metabolismoRESUMO
By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.
Assuntos
Conexinas/genética , Junções Comunicantes/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Conexinas/química , Conexinas/classificação , Conexinas/metabolismo , Junções Comunicantes/química , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Filogenia , Alinhamento de Sequência , Distribuição Tecidual , Xenopus laevisRESUMO
The multiple combinations of nAChR subunits identified in central nervous structures possess distinct pharmacological and physiological properties. A growing number of data have shown that compounds interacting with neuronal nAChRs have, both in vivo and in vitro, the potential to be neuroprotective and that treatment with nAChR agonists elicit long-lasting improving of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Epidemiological and clinical studies suggested also a potential neuroprotective/trophic role of (-)-nicotine in neurodegenerative disease, such as Alzheimer's and Parkinson's disease. Taken together experimental and clinical data largely indicate a neuroprotective/trophic role of nAChR activation involving mainly alpha7 and alpha4beta2 nAChR subtypes, as evidenced using selective nAChR antagonists, and by potent nAChR agonists recently found displaying efficacy and/or larger selective affinities than (-)-nicotine for neuronal nAChR subtypes. A neurotrophic factor gene regulation by nAChR signalling has been taken into consideration as possible mechanism involved in neuroprotective/trophic effects by nAChR activation and has evidenced an involvement of the fibroblast growth factor (FGF-2) gene as a target of nAChR signalling. These findings suggested that FGF-2 could be involved, according to the FGF-2 neurotrophic functions, in nAChR mechanisms mediating the neuronal survival, trophism and plasticity.
Assuntos
Encéfalo/fisiologia , Sobrevivência Celular/fisiologia , Fatores de Crescimento Neural/fisiologia , Fármacos Neuroprotetores , Receptores Nicotínicos/fisiologia , Animais , Fator 2 de Crescimento de Fibroblastos/fisiologia , Haplorrinos , Humanos , Neurônios/fisiologia , RatosRESUMO
The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Biomarcadores , Encéfalo/citologia , Mapeamento Encefálico , Junções Comunicantes/metabolismo , Imuno-Histoquímica , Masculino , Neurônios/citologia , Proteínas Nucleares/metabolismo , Parvalbuminas/metabolismo , Glândula Pineal/citologia , Glândula Pineal/metabolismo , Hipófise/citologia , Hipófise/metabolismo , RNA Mensageiro/metabolismo , Ratos , Proteína delta-2 de Junções ComunicantesRESUMO
Cx36 is the first mammalian member of a novel subgroup of the connexin family, characterized by a long cytoplasmic loop, a peculiar gene structure and a preferential expression in cell types of neural origin. In the present review we summarize the evidence in favour of its predominant expression in neuronal cells in the mammalian central nervous system, such as results from experiments with specific neurotoxins and co-localization of Cx36 mRNA and a neuronal marker. We also report a detailed description of Cx36 mRNA distribution in the rat and human central nervous system by in situ hybridization and, for each brain region, we correlate the novel findings with previous morphological or functional demonstrations of neuronal gap junctions in the same area.
Assuntos
Conexinas/genética , Proteínas do Olho/genética , Junções Comunicantes/fisiologia , Sinapses/fisiologia , Animais , Comunicação Celular/fisiologia , Junções Comunicantes/química , Expressão Gênica/fisiologia , Humanos , Mamíferos , Sinapses/química , Proteína delta-2 de Junções ComunicantesRESUMO
A growing number of data have shown that compounds interacting with neuronal nicotinic acetylcholine receptors (nAChRs) have, both in vivo and in vitro, the potential to be neuroprotective and that treatment with nAChR agonists elicit long-lasting improvement of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Epidemiological and clinical studies suggested also a potential neuroprotective/trophic role of (-)-nicotine in neurodegenerative disease, such as Alzheimer's disease and Parkinson's disease. This neuroprotective/trophic role of nAChR activation has been mainly mediated by alpha7 and alpha4beta2 nAChR subtypes, as evidenced using selective nAChR antagonists, and by potent nAChR agonists recently found displaying efficacy and/or larger selective affinities than (-)-nicotine for neuronal nAChR subtypes. A neurotrophic factor gene regulation by nAChR signalling has been taken into consideration as a possible mechanism involved in neuroprotective/trophic effects of nAChR activation and has given evidence that the fibroblast growth factor (FGF-2) gene is a target for nAChR signalling. These findings suggested that FGF-2 could be involved, in view of its neurotrophic functions, in nAChR mechanisms mediating neuronal survival, trophism and plasticity.
Assuntos
Fatores de Crescimento Neural/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Rat connexin-36 (Cx36) is the first gap junction protein shown to be expressed predominantly in neuronal cells of the mammalian central nervous system. As a prerequisite for studies devoted to the investigation of the possible role of this connexin in human neurological diseases, we report the cloning and sequencing of the human Cx36 gene, its chromosomal localization, and its pattern of expression in the human brain analyzed by radioactive in situ hybridization. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved (98% identity at the protein level with the mouse and rat Cx36 and 80% with the ortholog perch and skate Cx35), and that the gene structure is that typical of the Cx35/36 subgroup observed in the other species (presence of a single intron located within the coding region, 71 bp after the translation initiation site). The distribution of Cx36 in several regions of the human central nervous system is similar to that previously observed in rat brain. The most intense signal among the cerebral areas examined by in situ hybridization was observed in the inferior olivary complex, both in principal and accessory nuclei. A moderate labeling was also observed in several myelencephalic nuclei, in specific cells of the the cerebellar cortex, in a relatively large subpopulation of cells in the cerebral cortex, in the hilus of the dentate gyrus, and in the strata radiatum and oriens of hippocampal subfields. Moreover, labeled cells were revealed in all the lamina of the spinal cord gray matter. The chromosomal localization of the human Cx36 gene was determined by fluorescence in situ hybridization. The results allowed assignment of the gene to band 15q14, thus making it a possible candidate gene for a form of familial epilepsy previously linked to the same chromosomal band. The knowledge of the human Cx36 gene sequence, of its chromosomal localization, and of its pattern of expression opens new avenues for the analysis of its possible involvement in human genetic and acquired neuropathology.
Assuntos
Encéfalo/metabolismo , Cromossomos Humanos Par 15 , Conexinas/genética , Proteínas do Olho/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Conexinas/análise , Conexinas/metabolismo , Proteínas do Olho/análise , Proteínas do Olho/metabolismo , Feminino , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Especificidade de Órgãos , Iniciação Traducional da Cadeia Peptídica , Percas , Reação em Cadeia da Polimerase , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Rajidae , Medula Espinal/metabolismo , Proteína delta-2 de Junções ComunicantesRESUMO
Levels of mRNA for neurotrophins (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT-3; neurotrophin 4, NT-4) and their receptors (trkA, trkB, trkC) and for glial cell line-derived neurotrophic factor (GDNF) and its receptors (ret, GDNFR-alpha) were measured in rat thyroid tissue by ribonuclease protection assays. In thyroid tissue the NT-3 mRNA level was threefold lower and the NT-4 mRNA level sixfold higher than those detected in adult rat hippocampus, while BDNF mRNA was undetectable. Very low levels of mRNA for truncated trkB and trkC receptors and no catalytic trkA, trkB or trkC were found. In conclusion NT-3 and NT-4, but not the corresponding functional receptors, are expressed in the thyroid tissue. Therefore, it is unlikely that these factors serve a direct local autocrine or paracrine function in thyroid cell types, and a target-derived mode of action on neurons innervating the thyroid tissue is suggested. An opposite result has been found for the neurotrophic factor GDNF: thyroid tissue showed a high level of transcripts for the GDNF receptor subunits (GDNFR-alpha and Ret), while GDNF mRNA was undetectable. The in situ hybridization analysis of GDNFR-alpha and ret mRNA revealed an interesting difference in the cell distribution of these transcripts: ret mRNA is selectively expressed in a subpopulation of cells scattered in the follicular epithelium and in the interfollicular spaces, while GDNFR-alpha expression is more homogeneous and widespread, including the more abundant cell type of the thyroid gland: the follicular cell. Double-labeling in situ hybridization/immunocytochemistry experiments, with a specific marker (calcitonin), showed that parafollicular cells express ret but not GDNFR-alpha. This differential distribution of the GDNF receptor components (GDNFR-alpha and ret) may reflect a peculiar biological role in intercellular communication in the thyroid gland.
Assuntos
Proteínas de Drosophila , Fatores de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Glândula Tireoide/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurotrofina 3 , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro , Ratos , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator Neutrófico Ciliar , Receptor trkA , Receptor trkC , Receptores de Fator de Crescimento Neural/genética , Glândula Tireoide/ultraestruturaRESUMO
The present experiments were designed to extend previous work showing that acute intermittent (-)nicotine treatment upregulates the level of fibroblast growth factor-2 (FGF2) mRNA in several rat brain regions, by the use of the nicotinic acetylcholine receptor (nAChR) agonist ABT-594 with preferential selectivity for the alpha4beta2 nAChR subtype. ABT594 treatment led to a well-defined temporal and regional upregulation of FGF-2 mRNA. A double labelling analysis showed that the up-regulation of FGF-2 mRNA involves both neuronal and non-neuronal cells. The effects of ABT-594 on FGF-2 expression were antagonized by the preferential alpha4beta2 antagonist dihydrobetaerythroidine (DHbetaE), but not by alpha7 antagonist methyllycaconitine (MLA). In conclusion, FGF-2 mRNA levels can be increased in several brain regions upon alpha4beta2 nAChR activation, suggesting a therapeutic significance in neurodegenerative disorders.